Gu Lixiao, Chen Yuling, Wang Qingtao, Li Xiaojing, Mi Kaixia, Deng Haiteng
MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University , Beijing 100084, China.
Beijing Chaoyang Hospital, Capital Medical University , Beijing 100020, China.
J Proteome Res. 2015 Nov 6;14(11):4441-9. doi: 10.1021/acs.jproteome.5b00359. Epub 2015 Sep 29.
Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a Mycobacterium smegmatis sirtuin, MSMEG_5175, was overexpressed in a M. smegmatis strain mc(2)155 to generate an MSMEG_5175-overexpression strain (mc(2)155-MS5175) in the present study. The physiological aspects of mc(2)155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc(2)155 containing empty pMV261 plasmid (mc(2)155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc(2)155-pMV261 and mc(2)155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc(2)155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc(2)155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc(2)155-MS5175 as compared to those in mc(2)155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria.
烟酰胺腺嘌呤二核苷酸（NAD）依赖性脱乙酰酶（沉默调节蛋白）从原核生物到真核生物都高度保守。哺乳动物沉默调节蛋白的功能和调控已得到广泛研究，表明其在生物过程调控中发挥重要作用，而分枝杆菌沉默调节蛋白的功能研究较少。为了研究分枝杆菌中类沉默调节蛋白的功能，本研究在耻垢分枝杆菌mc(2)155菌株中过表达了一种耻垢分枝杆菌沉默调节蛋白MSMEG_5175，构建了MSMEG_5175过表达菌株（mc(2)155-MS5175）。对mc(2)155-MS5175菌株的生理特性进行了表征，结果显示与含有空pMV261质粒的mc(2)155（mc(2)155-pMV261）相比，它们的细胞内NAD水平较低，对异烟肼（INH）的抗性较高。进行了定量蛋白质组学分析，以确定mc(2)155-pMV261和mc(2)155-MS5175之间差异表达的蛋白质。在鉴定出的3032种蛋白质中，MSMEG_5175的过表达导致34种蛋白质上调和72种蛋白质下调，这些蛋白质涉及多种细胞过程，包括代谢激活、转录和翻译、抗氧化以及DNA修复。在mc(2)155-MS5175菌株中，过氧化氢酶过氧化物酶（KatG）的mRNA和蛋白质水平表达均下调，这表明细胞内NAD含量的降低和KatG表达的下调有助于mc(2)155-MS5175对INH的更高抗性。通过免疫沉淀和蛋白质组学分析相结合的方法，我们发现与mc(2)155-pMV261相比，mc(2)155-MS5175中有27种蛋白质的乙酰化水平降低，这表明这些蛋白质包括RNA聚合酶的β'亚基（rpoC）、核糖体蛋白和代谢酶是MSMEG_5175的底物。rpoC的乙酰化变化可能会影响其功能并导致全局基因转录的改变。综上所述，这些结果表明MSMEG_5175调节多种细胞过程导致分枝杆菌对INH的抗性增加，并为进一步深入研究分枝杆菌中蛋白质乙酰化的功能提供了有用的资源。
